Extracellular-signal regulated kinases 1 and 2 (ERK1/2) are the prototypical intracellular mitogen activated protein kinases (MAPK). ERK1/2 are activated by multiple growth factors and cytokines in many cell types. Studies in vitro implicate ERK1/2 in a myriad of cellular processes during proliferation, differentiation and apoptosis. ERK activity has both stimulatory and inhibitory roles in the differentiation of culture skeletal myotubes that vary with the stage of this protracted process. However, no gene targeting investigations on the role of ERK1/2 in developing muscle fibers in vivo have been reported to date. Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction (NMJ), the synapse between a motoneuron in the spinal cord and a skeletal muscle fiber. Agrin acts by binding and activating a receptor complex containing LDL receptor related protein 4 (Lrp4) and muscle specific kinase (MuSK). We reported recently that in cultured myotubes agrin induces ERK1/2 activation in an Lrp4/MuSK-dependent fashion and that this negatively regulates the ability of agrin to induce AChR clusters. Furthermore, our preliminary data show that loss of ERK2 in myofibers leads to defects in NMJ maintenance in young adult mice. In these animals, NMJs display fragmentation similar to than seen in NMJs of old mice or in mdx mice, which model Duchenne muscular dystrophy. The aim of this R21 application is to investigate the significance in vivo of ERK1/2 for neuromuscular synapse formation and maintenance and for skeletal myofiber differentiation and maturation. A strategy of conditional gene targeting that has recently bear fruit in other cells of the peripheral nervous system will be used to selectively inactivate Erk1/2 in skeletal muscle fibers. Results from the above experiments will clarify the in vivo role of ERK1/2 in agrin signaling at the NMJ, in particular, and in the development of skeletal muscle fibers, in general. In addition, they may provide new insights into the molecular mechanisms underlying normal aging, Duchenne muscular dystrophy and diseases caused by mutation of ERK1/2 downstream substrates expressed in muscle, such as ribosomal S6 kinase 2 (Rsk2), which is linked to Coffin-Lowry syndrome. PUBLIC HEALTH RELEVANCE: This project will investigate whether the protein kinases ERK1/2 have important roles in skeletal muscle differentiation and formation and maintenance of nerve-muscle connections in vivo. Results from these experiments may also be relevant for normal aging, Duchenne muscular dystrophy and Coffin-Lowry syndrome.